Natural killer (NK) cells are large granular lymphocytes of bone marrow origin that exhibit cytolytic activity against some tumor and virally infected cells in the absence of prior stimulation. We have previously identified murine 2B4 as an NK receptor expressed on a subset of T cells as well as on all murine NK cells. 2B4 is expressed early in NK cell development, before expression of other NK cell specific markers such as NK1.1 or Ly-49 family members. 2B4 is a member of the CD2 subfamily of the immunoglobulin superfamily and is expressed as 2 alternative splice variants. Litigation of 2B4 with mAb modulates target cell lysis and IFN-gamma production. Recently, we have shown CD48 to be the high affinity counter-receptor for 2B4. CD48 is widely expressed on cells of hematopoietic origin and is important in both T cell and B cell activation. The overall goal of this proposal is to investigate the role of 2B4 in the immune system, both as a NK cell receptor and as a counter- receptor for CD48. First, we will clone and characterize the human homologues of murine 2B4. To complement the murine 2B4 reagents we already have, we will generate monoclonal antibodies against 2B4, and soluble fusion proteins of 2B4 and CD48. Using these reagents, we will study the expression pattern of 2B4 in detail. Next, the role of 2B4 in the cytolytic function and IFN-gamma production of human NK cells will be analyzed. Furthermore, these reagents will allow us to study the role of 2B4-CD48 interactions on the regulation of CD48+ lymphocytes by 2B4+ cells. The effect of 2B4-CD48 interactions on B cells will be studied by analyzing their ability to provide (or block) co-stimulatory signals. This effect will be measured by proliferation and immunoglobulin production. Finally, the role of 2B4 in NK cell development and function will be established by using a 2B4 gene knockout mouse model. The experiments proposed in this project should provide a clearer understanding of the role of 2B4 in modulating various functions of human NK cells and may reveal new insights and open new avenues for cancer immunotherapy.